Method for rapidly preparing siRNA interference BLM helicase stable cell line

A technology of helicase and cell lines, which is applied in the field of rapidly obtaining siRNA interference BLM helicase stable cell lines, can solve the problems of time-consuming and laborious, high false positive rate, etc., and achieve the reduction of false positive rate, wide application range, and optimal screening effect of effect

Inactive Publication Date: 2018-07-10
GUIZHOU UNIV
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Problems solved by technology

[0004] The screening of traditional cell lines requires multiple rounds of resistance screening and dilution after transfection to obtain positive monoclonals, which is time-consuming and laborious, and the false positive rate is high

Method used

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  • Method for rapidly preparing siRNA interference BLM helicase stable cell line
  • Method for rapidly preparing siRNA interference BLM helicase stable cell line
  • Method for rapidly preparing siRNA interference BLM helicase stable cell line

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Embodiment Construction

[0034] The present invention will be further described below in conjunction with the examples, but not as a basis for limiting the present invention.

[0035] Embodiments of the invention

[0036] A method for rapidly obtaining siRNA interference BLM helicase stable cell line, the method is as follows:

[0037] 1) Design and synthesis of siRNA: According to the target sequence of the BLM helicase gene on NCBI, start from the start codon of transcription AUG according to the design principle of siRNA, search for the downstream AA sequence, and record the 19 nuclei adjacent to the 3' end of each AA Nucleotides are used as candidate siRNA target sites; DNA templates for the BLM helicase gene are synthesized according to the above design principles, and its sequence is BamH I restriction site, 21nt sense strand, 9nt loop structure, 21nt antisense strand, RNA Enzyme III transcription termination site (6 T), EcoR I restriction site, negative control sequence is BamH I restriction s...

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Abstract

The invention discloses a method for rapidly preparing a siRNA interference BLM helicase stable cell line. The method comprises 1) designing and synthesizing siRNA, 2) constructing a siRNA expressionvector, 3) cloning a transformation interference vector, 4) transfecting shRNA, 5) carrying out positive cell sorting through a flow cytometry through sorting fluorescent protein GFP-positive cells toa microwell plate containing a whole serum medium according to a way that one cell is sorted to one hole, and 6) culturing the sorted positive cells, extracting total RNAs of the cells, designing BLMgene primers, carrying out real-time fluorescence quantitative QRT-PCR detection on the mRNA expression level of the BLM gene, selecting a cell line silencing the BLM helicase and carrying out enlarging cultivation and cryopreservation. The method can fast and simply prepare a cell line, saves time and labor and has a low false positive rate.

Description

technical field [0001] The invention relates to a method for quickly obtaining a stable cell line of siRNA interference with BLM helicase, in particular a method for rapidly obtaining a stable cell line of siRNA interference with BLM helicase by using flow cytometry. Background technique [0002] BLM helicase (BLM) is an important member of the RecQ family of DNA helicases, involved in cellular metabolic processes such as DNA replication, repair, transcription, recombination, and telomere maintenance, and plays an important role in maintaining chromosome stability . Deletion of the BLM helicase can lead to BLM syndrome. BLM syndrome (BLM's syndrome, BS) is a rare autosomal recessive genetic disease. The clinical features of BS patients are severe growth retardation, learning disabilities, and immune deficiency. Studies have shown that BLM helicase is highly expressed in testis and thymus, and the mutation and high expression of BLM gene are closely related to tumorigenesis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/113C12N15/85
CPCC12N5/0693C12N9/14C12N15/113C12N15/85C12N2310/14C12Y306/04012
Inventor 许厚强刘忠伟罗霂榃陈福吴萍
Owner GUIZHOU UNIV
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